Environmental stresses, such as drought, increased salinity of soil, and extreme temperature, are major factors in limiting plant growth and productivity. The worldwide loss in yield of three major cereal crops, rice, maize (corn), and wheat due to water stress (drought) has been estimated to be over ten billion dollars annually. Drought and soil salinity are the most serious environmental stresses that limit plant growth and crop productivity (Boyer, “Plant Productivity and Environment,” Science, 218:443–448 (1982); Le Rudulier et al., “Molecular Biology of Osmoregulation,” Science, 224:1064–1068 (1984)). Of the 4,870 million hectares of agricultural land in the world, 930 million (19% of total) are salt-affected areas (FAO Quarterly Bulletin of Statistics, Vol. 9¾ (1996)). Moderate levels of salt content in the soil (such as 50 mM) cause a substantial decrease in the yield of crops. High levels of salt in the soil (higher than 100 or 150 mM) are not at all suitable for planting most cereal crops. Approximately 5.2% of the agricultural lands are under drought stress (FAO Quarterly Bulletin of Statistics, Vol. 9¾ (1996)), and the loss of crop yield is also very significant.
In practical terms, rice is the most important crop, because a high percentage of the world's population depends on it for their staple food. Together with wheat and corn, these three cereal crops constitute the major source of food and calories to feed the people. With an increase in population and a decrease in arable land, there is a real possibility of a food shortage by the year 2030. Therefore, it is essential to fully utilize plant biotechnology to improve plants and produce more food.
Breeding of stress-tolerant crop cultivars represents a promising strategy to tackle these problems (Epstein et al., “Saline Culture of Crops: A Genetic Approach,” Science, 210:399–404 (1980)). However, conventional breeding is a slow process for generating crop varieties with improved tolerance to stress conditions. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species are additional problems encountered in conventional breeding. Recent progress in plant genetic transformation and availability of potentially useful genes characterized from different sources make it possible to generate stress-tolerant crops using transgenic approaches (Tarczynski et al., “Stress Protection of Transgenic Tobacco by Production of the Osmolyte Mannitol,” Science, 259:508–510 (1993); Pilon-Smits et al., “Improved Performance of Transgenic Fructan-Accumulating Tobacco Under Drought Stress,” Physiol. Plant, 107:125–130 (1995)). Transformation of cereal plants with agronomically useful genes that increase tolerance to abiotic stress is one important way to minimize yield loss. For example, it would be highly desirable to produce transgenic rice plants that can give reasonable yield when grown in marginal or waste lands that contain relatively high levels of salt, such as 100–150 mM, in the soil.
Characterization and cloning of plant genes that confer stress tolerance remains a challenge. Genetic studies revealed that tolerance to drought and salinity in some crop varieties is principally due to additive gene effects (Akbar et al., “Breeding For Soil Stress,” In Progress in Rainfed Lowland Rice, International Rice Research Institute, manila, Philippines, pp. 263–272 (1986); Akbar et al., “Genetics of Salt Tolerance in Rice,” In Rice Genetics, International Rice Research Institute, Manila, Philippines, pp. 399–409 (1986)). However, the underlying molecular mechanism for the tolerance has never been revealed. Physiological and biochemical responses to high levels of ionic or nonionic solutes and decreased water potential have been studied in a variety of plants. Based on accumulated experimental observations and theoretical consideration, one suggested mechanism that may underlie the adaptation or tolerance of plants to osmotic stresses is the accumulation of compatible, low molecular weight osmolytes such as sugar alcohols, special amino acids, and glycine betaine (Greenway et al., “Mechanisms of Salt Tolerance in Nonhalophytes,” Annu. Rev. Plant Physiol., 31: 149–190 (1980); Yancey et al., “Living With Water Stress: Evolution of Osmolyte System,” Science, 217: 1214–1222 (1982)). In particular, proline level is known to increase in a number of plants and bacteria under drought or salt stress. Recently, a transgenic study has demonstrated that accumulation of the sugar alcohol mannitol in transgenic tobacco conferred protection against salt stress (Tarczynski et al., “Stress Protection of Transgenic Tobacco by Production of the Osmolyte Mannitol,” Science, 259:508–510 (1993)). Two recent studies using a transgenic approach have demonstrated that metabolic engineering of the glycine betaine biosynthesis pathway is not only possible but also may eventually lead to production of stress-tolerant plants (Holmstrom et al., “Production of the Escherichia coli Betaine-Aldehyde Dehydrogenase, An Enzyme Required for the Synthesis of the Osmoprotectant Glycine Betaine, in Transgenic Plants,” Plant J., 6:749–758 (1994); Rathinasabapathi et al., “Metabolic Engineering of Glycine Betaine Synthesis: Plant Betaine Aldehyde Dehydrogenases Lacking Typical Transit Peptides are Targeted to Tobacco Chloroplasts Where they Confer Betaine Aldehyde Resistance,” Planta, 193:155–162 (1994)).
In addition to metabolic changes and accumulation of low molecular weight compounds, a large set of genes is transcriptionally activated which leads to accumulation of new proteins in vegetative tissue of plants under osmotic stress conditions, including the late embryogenesis abundant (LEA) family, dehydrines, and COR47 (Skriver et al., “Gene Expression in Response to Abscisic Acid and Osmotic Stress,” Plant Cell, 2:503–512 (1990); Chandler et al., “Gene Expression Regulated by Abscisic Acid and its Relation to Stress Tolerance,” Annu. Rev. Plant Physiol. Plant Mol. Biol., 45:113–141 (1994)). The expression levels of a number of genes have been reported to be correlated with desiccation, salt, or cold tolerance of different plant varieties of the same species. It is generally assumed that stress-induced proteins might play a role in tolerance, but the functions of many stress-responsive genes are unknown.
Elucidating the function of these stress-responsive genes and enzymes involved in the biosynthesis of stress-induced osmolytes will not only advance the understanding of plant adaptation and tolerance to environmental stresses, but also may provide important information for designing new strategies for crop improvement (Chandler et al., “Gene Expression Regulated by Abscisic Acid and its Relation to Stress Tolerance,” Annu. Rev. Plant Physiol. Plant Mol. Biol., 45:113–141 (1994)).
In recent years, different stress-tolerant transgenic plants have been obtained (Tarczynski et al., “Stress Protection of Transgenic Tobacco by Production of Osmotic Mannitol,” Science, 259:508–510 (1993); Shen et al., “Increased Resistance to Oxidative Stress in Transgenic Plants by Targeting Mannitol Biosynthesis to Chloroplasts,” Plant Physiol., 113:1177–1183 (1997); Kishor et al., “Overexpression of Δ1-pyrroline-5-carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants,” Plant Physiol., 108:1387–1394 (1995); Pilon-Smits et al., “Improved Performance of Transgenic Fructan-Accumulating Tobacco Under Drought Stress,” Plant Physiol., 107:125–130 (1995); Holmstrom et al., “Drought Tolerance on Tobacco,” Nature, 379:683–684 (1996); Xu et al., “Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice,” Plant Physiol., 110:249–257 (1996); Nomura et al., Synechococcus sp. PPC 7942 Transformed with E. coli bet Genes Produces Glycine Betaine from Choline and Acquires Resistance to Salt Stress,” Plant Physiol., 107:703–708 (1995); Hayashi et al., “Transformation of Arabidopsis thaliana with codA Gene for Choline Oxidase: Accumulation of Glycine-Betaine and Enhanced Tolerance to Salt and Cold Stress,” Plant J., 12:133–142 (1997); Sheveleva et al., “Increased Salt and Drought Tolerance by D-ononitol Production in Transgenic Nicotiana tabacum L.,” Plant Physiol., 115:1211–1219 (1997)) by producing either a low molecular weight osmoprotectant (such as glycine betaine, mannitol, inositol, proline, fructan, trehalose, or D-ononitol) or a late embryogenesis abundant (LEA) protein. In transgenic tobacco transformed with the A1-pyrroline-5-carboxylate synthetase cDNA (p5cs cDNA), it was found that proline accumulation was correlated with tolerance to drought and salinity stresses in plants. Overproduction of proline also enhanced root biomass and flower development in transgenic tobacco under drought-stress conditions (Kishor et al., “Overexpression of Δ1-pyrroline-5-carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants,” Plant Physiol., 108:1387–1394 (1995)). Proline is believed to be involved in osmotic adjustment, primarily as a cytoplasmic solute (Voetberg et al., “Growth of the Maize Primary Root at Low Water Potentials. III. Role of Increased Proline Deposition in Osmotic Adjustment,” Plant Physiol., 96:1125–1130 (1991)), as an osmoprotectant (Kishor et al., “Overexpression of Δ1-pyrroline-5-carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants,” Plant Physiol., 108:1387–1394 (1995)), and as a hydroxy radical scavenger (Smimoff et al., “Hydroxyl Radical Scavenging Activity of Compatible Solutes,” Phytochemistry, 28:1057–1060 (1989)). Proline has also been reported to play a role in protecting enzymes from denaturation (Nikolopoulos et al., “Compatible Solutes and in vitro Stability of Salsola soda Enzymes: Proline Incompatibility,” Phytochemistry, 30:411–413 (1991)) and stabilizing the machinery of protein synthesis (Kadpal et al., “Alterations in the Biosynthesis of Proteins and Nucleic Acids in Finger Millet (Eleucine coracana) Seedlings During Water Stress and the Effect of Proline on Protein Biosynthesis,” Plant Science, 40:73–79 (1985)). Some or all of the presumed functions may contribute to osmotolerance of transgenic plants that overproduce proline. In most of the above-noted reports, tobacco (a dicot) was used as the model plant. Since dicots and monocots are quite different in their physiology, morphology, and, perhaps, response to abiotic stresses as well, it is important to study how overproduction of proline affects a major monocot cereal plant, such as rice, in response to stresses.
In addition, under normal environmental conditions, overproduction of the above-noted compounds or proteins will need extra energy and building blocks and may hamper the normal growth of plants. Thus, it is desirable to generate transgenic plants which synthesize a high level of an osmoprotectant or a protein only under stress conditions.
The phytohormone abscisic acid (ABA) is thought to mediate physiological processes in response to osmotic stress in plants (King, “Abscisic Acid in Developing Wheat Grains and its Relationship to Grain Growth and Maturation,” Planta, 132:43–51(1976); Jones et al., “The Effect of Abscisic Acid on Cell Turgor Pressures, Solute Content, and Growth of Wheat Roots,” Planta, 170:257–262 (1987)). Water stress by NaCl or dehydration can cause endogenous ABA levels to increase in plant tissues (Henson, “Effects of Atmospheric Humidity on Abscisic Acid Accumulation and Water in Leaves of Rice (Oryza sativa L.),” Ann. Bot., 54:569–582 (1984); Jones et al., “The Effect of Abscisic Acid on Cell Turgor Pressures, Solute Content, and Growth of Wheat Roots,” Planta, 170:257–262 (1987)). Mundy et al., “Abscisic Acid and Water-Stress Induce the Expression of a Novel Rice Gene,” The EMBO J., 7:2279–2286 (1988) found that ABA controls the accumulation of specific mRNAs and proteins both from developmental studies with seeds and physiological studies with water stressed tissues. Specific genes are expressed under stress conditions and can also be induced in unstressed tissues by the application of exogenous ABA (Singh et al., “Hormonal Regulation of Protein Synthesis Associated with Salt Tolerance in Plant Cell,” Proc. Natl. Acad. Sci. USA, 84:739–743 (1987); Gomez et al., “A Gene Induced by the Plant Hormone Abscisic Acid in Response to Water Stress Encodes a Glycine-rich Protein,” Nature, 334:262–264 (1988); Mundy et al., “Abscisic Acid and Water-Stress Induce the Expression of a Novel Rice Gene,” The EMBO J., 7:2279–2286 (1988); Chandler et al., “Gene Expression Regulated by Abscisic Acid and its Regulation to Stress Tolerance,” Annu. Rev. Plant Physiol.& Mol. Biol. 45:113–114 (1994)).
In addition to the studies on the physiological roles of ABA, efforts are being made to investigate the molecular mechanism of ABA action, including the definition of ABA-response elements (ABREs) and the trans-acting factors that interact with ABREs. It was reported that a 75-bp fragment of the ABA-inducible wheat Em gene, when fused to a truncated CaMV 35S promoter, conferred a more than 10-fold ABA induction of GUS activity in rice protoplasts (Guiltinan et al., “A Plant Leucine Zipper Protein that Recognizes an Abscisic Acid Response Element,” Science, 250:267–271 (1990)). Guiltinan et al. also found a leucine-zipper DNA binding protein, EmBP-1, which binds the ABRE sequence (CACGTGGC) in this 75-bp region. Transient assays in rice protoplasts revealed a 40-bp ABA-responsive fragment in the rice rab 16B promoter (Ono et al., “The rab 16B Promoter of Rice Contains Two Distinct Abscisic Acid-Responsive Elements,” Plant Physiol., 112:483–491 (1996)). Two separate ABREs, motif I and motif III, are required for ABA induction; however, each can substitute for the other. The 40-bp-fragment-containing motif I fused to a truncated CaMV 35S promoter showed an approximate 4- to 5-fold induction by ABA (Ono et al., “The rab 16B Promoter of Rice Contains Two Distinct Abscisic Acid-Responsive Elements,” Plant Physiol., 112:483–491 (1996)). The ABREs are very similar to the G-box, which, as has been pointed out by Guiltinan et al., “A Plant Leucine Zipper Protein that Recognizes an Abscisic Acid Response Element,” Science, 250:267–271 (1990), is present in some genes that are responsive to other environmental and physiological stimuli such as light (Giuliano et al., “An Evolutionarily Conserved Protein Binding Sequence Upstream of a Plant Light-Regulated Gene,” Proc. Natl. Acad. Sci. USA, 85:7089–7093 (1988)) and auxin (Liu et al., “Soybean GH3 Promoter Contains Multiple Auxin-Inducible Elements,” Plant Cell, 6:645–657 (1994)).
Studies on the promoter of the barley ABA-responsive HVA22 gene indicate that G-box sequences are necessary but not sufficient for ABA response (Shen et al., “Functional Dissection of an Abscisic Acid (ABA)-Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containing a G-Box and Novel cis-Acting Element,” The Plant Cell, 7:295–307 (1995)). Instead, an ABA-responsive complex consisting of a G-box, namely, ABRE3, and a novel coupling element, CE1, is sufficient for high-level ABA induction. The results of linker-scan analyses and gain-of-function studies showed that the 49-bp ABA- response complex (ABRC 1) is the minimal sequence governing high-level ABA induction. A similar investigation on ABA induction of a barley late embryogenesis abundant (LEA) gene HVA1 (Shen et al., “Modular Nature of Abscisic Acid (ABA) Response Complexes: Composite Promoter Units that are Necessary and Sufficient for ABA Induction of Gene Expression in Barley,” The Plant Cell, 8:1107–1119 (1996)) was conducted. Shen et al. found that the ABRC3 of this gene consists of a 10-bp element with an ACGT core (A2) and a sequence directly upstream, named CE3. Only one copy of this ABRC3 is sufficient to confer ABA induction when fused to a minimal promoter (Amy64). Thus, two types of ABRCs were reported by Shen et al., “Functional Dissection of an Abscisic Acid (ABA)-Inducible Gene Reveals Two Independent ABA-Responsive Complexes Each Containing a G-Box and Novel cis-Acting Element,” The Plant Cell, 7:295–307 (1995) and Shen et al., “Modular Nature of Abscisic Acid (ABA) Response Complexes: Composite Promoter Units that are Necessary and Sufficient for ABA Induction of Gene Expression in Barley,” The Plant Cell, 8:1107–1119 (1996), namely, ABRC1, consisting of ABRE3 and CE1 from HVA22 gene, and ABRC3, composed of CE3 and A2 from HVA1 gene.
The present invention is directed to producing transgenic cereal plants with improved water stress and salt stress tolerance.